The 310-helix is defined by intramolecular H-bonds between amino acid residues placed at positions i and i+3, and is an important structural motif in peptides and proteins. It plays important roles in many different biological recognition processes. It is the biologically active conformation of many pharmacologically interesting peptides/peptidomimetics. In addition, many protein segments mediating physiologically or pathophysiologically important interactions between biomolecules (e.g. two proteins or a peptide with a protein) adopt a 310-helical conformation.
Two examples are the interactions between aquaporin-4 (AQP4) and aquaporin-4 and between AQP4 and the antibody NMO-IgG, the latter being important in the pathophysiology of the multiple sclerosis like disease neuromyelitis optica (NMO).
The concept of introducing conformational constraints in peptides which stabilize their biologically active secondary structure has attracted a lot of interest as a way to improve the pharmacological properties of peptides. In particular, this concept has been applied to α-helical peptides and protein segments. Examples include peptides with intramolecular H-bond surrogates1 and so-called stapled peptides, the latter deriving helix stabilization from side chain-to-side chain hydrophobic interactions,2 salt bridges,3 disulfide bridges,4 lactams5 and metathesis derived hydrocarbon bridges.6-8 Significantly, hydrocarbon stapling of α-helical peptides has resulted in a number of compounds with clinical potential, e.g. against cancer.9 Recently, hydrocarbon stapling has also been successfully applied to 314-helical β-peptides,10 extending its range of applicability beyond α-peptides.
The 310-helix, which is defined by intramolecular i→i+3 H-bonds, is an important structural element in proteins, peptide antibiotics known as peptaibols,11 and many biological recognition processes, as well as a postulated intermediate structure in protein folding.12 
Over the last decade the predominant water channel in the mammalian brain, aquaporin-4 (AQP4), has emerged as an important target for treatment of brain edema after stroke or trauma.13-16 The present inventors considered the development of selective inhibitors of AQP4 based on side chain-to-side chain cyclised 310-helical analogues of the Pro138-Gly144 segment of human AQP4,17 which has been postulated to mediate adhesive interactions between two AQP4 tetramers.18-20 
Examples of i→i+3 and i→i+4 side chain-to-side chain crosslinking in 310-helical peptides by Glu-Lys lactam formation,21 ferrocenedicarboxylic acid Lys diamides,22 photoinduced 1,3-dipolar cycloaddition,23 metathesis derived hydrocarbon bridges,17,24,25 and a p-phenylenediacetic acid bridge26 between two α,α-disubstituted 4-aminopiperidine-4-carboxylic acid (Api) residues have been reported. However, only two studies25,26 have provided atomic resolution detail of the effect of cyclization on helix regularity, i.e. on backbone dihedral angles and H-bond lengths, and very little23 is known on how cyclization/stapling affects the thermal stability of the 310-helix.
In the first X-ray crystallographic study25 of the effect of side chain-to-side chain cyclization in a 310-helical peptide it was observed that the backbone is distorted by an i→i+3 metathesis derived olefinic bridge, resulting in the breakage of one intramolecular H-bond, thus disrupting the 310-helix. The p-phenylenediacetic acid bridge on the other hand appears to afford a highly regular Api/Aib based 310-helix.26 However, α,α-disubstituted amino acids like Aib and N-acylated Api are generally hydrophobic and have a tendency to distort the dihedral angles of neighbouring monosubstituted, proteinogenic residues away from ideality.21,25,27 Hence, alternative methodology for side chain-to-side chain crosslinking of monosubstituted residues, which are expected to be better tolerated in the context of a helical peptide primarily consisting of the proteinogenic amino acids, which does not significantly distort the regularity of the 310-helix, is highly desirable. If, at the same time, the crosslinking provides thermal stabilization of the 310-helix and results in a more hydrophilic bridge, thus increasing the aqueous solubility of the stapled peptide, such a methodology could potentially have broad utility to the study and modulation of biologically important recognition processes involving 310-helical peptides and protein segments.
There has been an explosion of interest in click chemistry28 in recent years, exemplified by the highly popular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.29-32 This reaction has been successfully applied to i→i+4 side chain-to-side chain cyclization in an α-helical peptide33,34 and i→i+3 cyclization in peptoids (peptides composed of N-substituted glycines).35 The high functional group tolerance of the CuAAC reaction, the very large dipole moment (˜5D)36 and the relatively high resistance to metabolic degradation37,38 of the 1,2,3-triazole moiety make 310-helical peptides with a side chain-to-side chain triazole bridge highly interesting objects of study.
The present inventors have installed an i→→i+3 constraint by side chain-to-side chain CuAAC between two monosubstituted residues in the context of a 310-helical Aib rich peptide and examined in detail the effect of cyclization on helix regularity and on helix stability. To allow a direct comparison with the results for the i→i+3 hydrocarbon bridge, two octapeptides 21 (Scheme 1) and 23 (Scheme 2) with the reactive/crosslinked residues in the same Aib rich context as the olefinic peptides of Boal et al25 were chosen as synthetic targets.
The inventors provide the first X-ray structural investigation of a (α- or 310-) helical peptide after stapling by CuAAC or with a triazole derived conformational constraint, the first systematic thermodynamic and computational analysis of any stapled 310-helical peptide and the first 2D IR structural investigation of a helical peptide with a conformational constraint installed. Surprisingly perhaps, given the widespread interest in the CuAAC reaction, this study will also afford what appears to be the first crystal structure of a difunctional azide-alkyne compound.